Field of the Invention
The present invention relates generally to the fields of gene biochemical pharmacology and drug discovery. More specifically, the present invention relates to method of screening for a compound that inhibits formation of an organism""s 5xe2x80x2 mRNA cap structure.
Description of the Related Art
Processing of eukaryotic mRNA in vivo is coordinated temporally and physically with transcription. The earliest event is the modification of the 5xe2x80x2 terminus of the nascent transcript to form the cap structure m7GpppN. The cap is formed by three enzymatic reactions: (i) the 5xe2x80x2 triphosphate end of the nascent RNA is hydrolyzed to a diphosphate by RNA 5xe2x80x2 triphosphatase; (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase: and (iii) the GpppN cap is methylated by AdoMet:RNA (guanine-N7) methyltransferase [1].
RNA capping is essential for cell growth. Mutations of the triphosphatase, guanylyltransferase, or methyltransferase components of the yeast capping apparatus that abrogate catalytic activity are lethal in vivo [2-12]. Genetic and biochemical experiments highlight roles for the cap in protecting mRNA from untimely degradation by cellular 5xe2x80x2 exonucleases [13] and in recruiting the mRNA to the ribosome during translation initiation [14].
The physical and functional organizations of the capping apparatus differ in significant respects in fungi, metazoans, protozoa, and viruses. Hence, the cap-forming enzymes are potential targets for antifungal, antiviral, and antiprotozoal drugs that would interfere with capping of pathogen mRNAs, but spare the mammalian host capping enzymes. A plausible strategy for drug discovery is to identify compounds that block cell growth contingent on pathogen-encoded capping activities without affecting the growth of otherwise identical cells bearing the capping enzymes of the host organism. For this approach to be feasible, the capping systems of interest must be interchangeable in vivo.
The architecture of the capping apparatus differs between metazoans, fungi, protozoa, and DNA viruses. Metazoan species encode a two-component capping system consisting of a bifunctional triphosphatase-guanylyltransferase polypeptide (named Mce1p in the mouse and Hce1p in humans) and a separate methyltransferase polypeptide (Hem1p in humans) [6, 9, 15-22]. The budding yeast Saccharomyces cerevisiae encodes a three component system consisting of separate triphosphatase (Cet1p). guanylyltransferase (Ceg1p), and methyltransferase (Abd1p) gene products [7, 10, 11, 23]. In yeast, the triphosphatase (Cet1p) and guanylyltransferase (Ceg1p) polypeptides interact to form a heteromeric complex [11], whereas in mammals, autonomous triphosphatase and guanylyltransferase domains are linked in cis within a single polypeptide (Mce1p) [18].
Vacciniavirus capping enzyme is a multifunctional protein that catalyzes all three reactions. The triphosphatase, guanylyltransferase, and methyltransferase active sites are arranged in a modular fashion within a single polypeptidexe2x80x94the Vaccinia D1 protein [24-30]. Other DNA viruses encode a subset of the capping activities; e.g., baculoviruses encode a bifunctional triphosphatase-guanylyltransferase (LEF-4) and Chlorella virus PBCV-1 encodes a monofunctional guanylyltransferase [31-33]. The guanylyltransferase and methyltransferase domains are conserved between DNA viruses, fungi, and metazoans. In contrast, the triphosphatase components are structurally and mechanistically divergent.
RNA Guanylyltransferasexe2x80x94Transfer of GMP from GTP to the 5xe2x80x2 diphosphate terminus of RNA occurs in a two-step reaction involving a covalent enzyme-GMP intermediate [34]. Both steps require a divalent cation cofactor, either magenesium or manganese.
E+pppG E-pG+PPi (i) 
E-pG+ppRNA GpppRNA+E (ii) 
The GMP is covalently linked to the enzyme through a phosphoamide (Pxe2x80x94N) bond to the epsilon-amino group of a lysine residue within a conserved KxDG element (motif I) found in all known cellular and DNA virus-encoded capping enzymes (FIG. 1). Five other sequence motifs. (III, IIIa, IV, and VI) are conserved in the same order and with similar spacing in the capping enzymes from fungi, metazoans, DNA viruses, and trypanosomes (FIG. 1) [35].
Hakansson et al. [36] have determined the crystal structure of the Chlorella virus capping enzyme in the GTP-bound state and with GMP bound covalently. The protein consist of a larger N-terminal domain (domain 1, containing motifs I, III, IIIa, and IV) and a smaller C-terminal domain (domain 2, containing motif VI) with a deep cleft between them. Motif V bridges the two domains. Motifs I, III, IIIa, and V form the nucleotide binding pocket. The crystal structure reveals a large conformational change in the GTP-bound enzyme, from an xe2x80x9copenxe2x80x9d to a xe2x80x9cclosedxe2x80x9d state that brings motif VI into contact with the beta and gamma phosphates of GTP and reorients the phosphates for in-line attack by the motif I lysine. When the crystal is soaked in maganese, guanylyltransferase reaction chemistry occurs in crystallo and the covalent enzyme-GMP intermediate is formed. However, only the enzyme in the closed conformation is reactive.
Identification of essential enzymic functional groups has been accomplished by site-directed mutagenesis of Ceg1p, the RNA guanylyltransferase of Saccharomyces cerevisiae. The guanylyltransferase activity of Ceg1p is essential for cell viability. Hence, mutational effects on Ceg1p function in vivo can be evaluated by simple exchange of mutant CEG1 alleles for the wild type gene. The effects of alanine substitutions for individual amino acids in motifs I, III, IIIa, IV, V, and VI [2, 5, 6] have been examined. Sixteen residues were defined as essential (denoted by asterisks in FIG. 1) and structure-activity relationships at these positions were subsequently determined by conservative replacements. Nine of the essential Ceg1p side chains correspond to moieties which, in the Chlorella virus capping enzyme crystal structure, make direct contact with GTP (arrowheads in FIG. 2). These include: the motif I lysine nucleophile which contacts the alpha-phosphate of GTP; the motif I arginine and motif III glutamate, which contact the 3xe2x80x2 and 2xe2x80x2 ribose hydroxyls, respectively: the motif III phenylalanine, which stacks on the guanine base; the two motif V lysines, which contact the alpha-phosphate; the motif V aspartate, which interacts with the beta-phosphate: the motif VI arginine that interacts with the beta-phosphate; and the motif VI lysine, which contacts the gamma-phosphate of GTP [6, 36].
On the basis of sequence conservation outside motif I, III, IIIa, IV, V, and VI, the capping enzymes of fungi (S. cerevisiae. S. pombe. C. albicans,), metazoans (C. elegans and mammals) and Chlorella virus can be grouped into a discrete subfamily [6, 37]. The sequence alignment in FIG. 2 highlights two motifs that are present in these capping enzymes, but not in the poxvirus enzymes, which can be designated motif P and motif Vc. In the Chlorella virus capping enzyme, motif P forms one wall of the guanosine binding pocket of domain 1 [36]. Motif Vcxe2x80x94(K/R)I(I/V)ECxe2x80x94is situated between motifs V and VI in domain 2. The glutamate residue of motif Vc is essential for the activity of the fungal guanylyltransferase Ceg1p [37].
RNA Triphosphatasexe2x80x94There are at least two mechanistically and structurally distinct classes of RNA 5xe2x80x2 triphosphatases: (i) the divalent cation-dependent RNA triphosphatase/NTPase family (exemplified by yeast Cet1p, baculovirus LEF-4 and vaccinia D1), which require three conserved collinear motifs (A, B, and C) for activity [12, 28, 31, 32], and (ii) the divalent cation-independent RNA triphosphatases, e.g., the metazoan cellular enzymes and the baculovirus enzyme BVP [15, 17, 38-40], which require the HCxAGxGR(S/T)G phosphate-binding motif. The existence of additional classes of RNA 5xe2x80x2-triphosphatases is likely, given that the candidate capping enzymes of several RNA viruses and trypanosomatid protozoa lack the defining motifs of the two known RNA triphosphatase families [41, 42]. Hence, the triphosphatase components of the capping apparatus provide attractive targets for the identification of specific antifungal, antiviral, and antiprotozoal drugs that will block capping of pathogen mRNAs, but spare the mammalian host enzyme.
Mammalian RNA Triphosphatasexe2x80x94Metazoan capping enzymes consist of an N-terminal RNA triphosphatase domain and a C-terminal guanylyltransferase domain. In the 496-amino acid mouse enzyme Mce1p, the two catalytic domains are autonomous and nonoverlapping [18]. The metazoan RNA triphosphatase domains contain a (I/V)HCxAGxGR(S/T)G signature motif initially described for the protein tyrosine phosphatase/dual-specificity protein phosphate enzyme family. These enzymes catalyze phosphoryl transfer from a protein phosphomonoester substrate to the thiolate of the cysteine of the signature motif to form a covalent phosphocysteine intermediate, which is then hydrolyzed to liberate phosphate (FIG. 3). The metazoan capping enzymes hydrolyze the phosphoanhydride bond between the beta and gamma phosphates of triphosphate-terminated RNA; they are not active on nucleoside triphosphates. The conserved cysteine of the signature motif is essential for RNA triphosphatase function [11, 15, 38, 39]. A characteristic of the cysteine-phosphatases is their lack of a requirement for a divalent cation cofactor.
The N-terminal portion of Mce1p from residues 1-210 is an autonomous RNA triphosphatase domain [18]. Recombinant Mce1(1-210)p has been expressed in bacteria and purified to near-homogeneity. Mce1(1-210)p sediments in a glycerol gradient as a discrete peak of 2.5 S. indicating that the domain is monomeric in solution. The RNA triphosphatase activity of Mce1(1-210)p can be assayed by the release of 32Pi from xcex332P-labeled poly(A). A kinetic analysis showed that the initial rate of Pi release was proportional to enzyme concentration. Mce1(1-210)p hydrolyzed 1.2 to 2 molecules of Pi per enzyme per second at steady state. RNA triphosphatase activity was optimal in 50 mM Tris buffer at pH 7.0 to 7.5. Activity was optimal in the absence of a divalent cation and was unaffected by EDTA. Inclusion of divalent cations elicited a concentration dependent inhibition of RNA triphosphatase activity. 75% inhibition was observed at 0.5 mM MgC12 or MnC12. Mce1(1-210) did not catalyze release of 32Pi from [xcex332P]ATP.
Metal-dependent RNA Triphosphatasesxe2x80x94The RNA triphosphatases of S. cerevisiae and DNA viruses are structurally and mechanistically unrelated to the metazoan RNA triphosphatases. The vaccinia virus RNA triphosphatase depends absolutely on a divalent cation cofactor. Vaccinia triphosphatase displays broad specificity in its ability to hydrolyze the gamma phosphate of ribonucleoside triphosphates, deoxynucleoside triphosphates, and triphosphate-terminated RNAs [43, 44]. The NTPase and RNA triphosphatase reactions occur at a single active site within an 545-amino acid N-terminal domain of vaccinia capping enzyme that is distinct from the guanylyltransferase active site [27-30]. The vaccinia RNA triphosphatase is optimal with magnesium, is 12% as active in manganese, and is inactive with cobalt [43]. In contrast, the vaccinia NTPase is fully active with cobalt, manganese, or magnesium [43, 44]. Baculovirus LEF-4 hydrolyzes the g phosphate of RNA and NTPs: the LEF-4 NTPase is activated by manganese or cobalt, but not by magnesium [31].
The yeast RNA 5xe2x80x2-triphosphatase Cet1p also hydrolyzes the gamma phosphate of nucleoside triphosphates [12]. The NTPase of Cet1p is activated by manganese and cobalt. This is a property shared with the triphosphatase components of the vaccinia D1 and baculovirus LEF-4 capping enzymes. Recent studies illuminate a common structural basis for metal-dependent catalysis by these enzymes. The metal-dependent RNA triphosphatases share 3 collinear sequence motifs, designated A, B, and C (FIG. 4). These are present in yeast Cet1p, in the Cet1p homolog from yeast Candida albicans, in the triphosphatase-guanylyltransferase domains of the vaccinia virus. Shope fibroma virus, molluseum contagiosum virus, and African swine fever virus capping enzymes, and in baculovirus LEF-4. Mutational analysis identified several residues within these motifs that are essential for the RNA triphosphatase and ATPase activities of vaccinia virus capping enzyme: the essential residues include two glutamates in motif A, an arginine in motif B, and two glutamates in motif C [28]. Alanine substitutions at any of these positions in the vaccinia capping enzyme reduced phosphohydrolase specific activity by 2 to 3 orders of magnitude. These 5 residues may comprise part of the triphosphatase active site. All five residues essential for vaccinia triphosphatase activity are conserved in LEF-4 and Cet1p. Mutations of the equivalent residues of Cet1p result in loss of triphosphatase activity [12].
Physical Association of the Triphosphatase and Guanylyltransferase Components of the Capping Apparatusxe2x80x94Yeast and mammals use different strategies to assembly a bifunctional enzyme with triphosphatase and guanylyltransferase activities. In yeast, separate triphosphatase (Cet1p and guanylyltransferase (Ceg1p) enzymes interact to form a heteromeric complex, whereas in mammals, autonomous triphosphatase and guanylyltransferase domains are linked in cis within a single polypeptide (Mce1p).
Cet1p and Ceg1p Form a Heterodimeric Capping Enzyme Complex In Vitroxe2x80x94The native size of purified recombinant yeast RNA triphosphatase Cet1p was gauged by glycerol gradient sedimentation. Cet1p sedimented as a single component of 4.3 S. The yeast guanylyltransferase Ceg1p sediments similarly in a glycerol gradient. Yet, when equal amounts of recombinant Cet1p and Ceg1p were mixed in buffer containing 0.1 M NaC1 and the mixture was analyzed by glycerol gradient sedimentation, the two proteins, as well as the triphosphatase and guanylyltransferase activities, cosedimented as a single discrete peak of 7.5 S. Thus, Ceg1p and Cet1p interact in vitro to form a heteromeric complex [11]. Cet1p does not form a complex with the structurally homologous RNA guanylyltransferase domain of the mouse capping enzyme. Recombinant mouse guanylyltransferase. Mce1(211-597)p, was purified to homogeneity, mixed with Cet1or with a buffer control, and then subjected to sedimentation analysis in parallel with the Cet1xe2x80x94Ceg1 mixtures. The 45 kDa Mce1(211-597) protein alone sedimented as a single monomeric peak [18]. Sedimentation of the Mce1(211-597)p plus Cet1p mixture revealed no shift in the distribution of the mouse guanylyltransferase or the yeast triphosphatase to a more rapidly sedimenting from [11]. Hence, yeast RNA triphosphatase forms a heteromeric complex in vitro with yeast guanylyltransferase, but not with the mammalian enzyme. Subsequent studies of the Candida albicans RNA triphosphatase (named CaCet1p) showed that it could interact with S. cerevisiae guanylyltransferase Ceg1p in vivo as gauged by a two-hybrid reporter assay. [45].
Cet1pxe2x80x94Cet1p Heterodimerization is Essential In Vivoxe2x80x94Truncated proteins Cet1(201-549)p and Cet1(246-549)p were expressed in bacteria and purified from soluble bacterial lysates by Ni-agarose and phosphocellulose column chromatography. Purified Cet1(201-549)p and Cet1(246-549)p catalyzed the release of 32Pi from xcex332P-labeled triphosphate-terminated poly(A) or [xcex332P]ATP with the same specific activity as full-length Cet1p [11]. The CET1(201-259) gene in single copy was functional in vivo in supporting yeast cell growth [11]. The finding that the CET1(246-549) gene on a CEN plasmid could not support cell growth, even though the gene product has full RNA triphosphatase activity in vitro, suggests that the catalytic activity of Cet1p, though essential for cell growth (see below), may not suffice for Cet1p function in vivo. Glycerol gradient analysis showed that Cet1(201-549)p by itself sedimented as a discrete species of xcx9c4.1 S. When Cet1(201-549)p was mixed with Ceg1p and the mixture was analyzed by glycerol gradient sedimentation, the two proteins cosedimented as a 6.8 S heteromeric complex [11]. The more extensively truncated Cet1(246-549)p did not sediment as a discrete species like full-sized Cet1p and Cet1(201-549)p. Rather, most of the Cet1(246-549)p sedimented as a high molecular weight oligomer (xcx9c13 S) that retained RNA triphosphatase activity. When a mixture of Cet1(246-549)p and Ceg1p was sedimented most of the Cet1(246-549)p remained aggregated; only a minor fraction of the input Ceg1p was shifted to the size expected for the heteromeric complex. Deletion from residues 201-245 (which results in loss of function in vivo despite retention of triphosphatase activity in vitro) affects the interaction of Cet1p with Ceg1p.
The implication of these data is that the interaction of Cet1p with Ceg1p is essential for yeast cell growth. Pharmacological interference with Ceg1pxe2x80x94Cet1p heterodimerization is a potential mechanism for blocking gene expression in fungi without impacting on mammalian cells.
Cap Methyltransferasexe2x80x94The enzyme RNA (guanine-N7-) methyltransferase (referred to hereafter as cap methyltransferase) catalyzes the transfer of a methyl group from AdoMet to the GpppN terminus of RNA to produce m7GpppN-terminated RNA and AdoHey [1]. The Saccharomyces cerevisiae cap methyltransferase is the product of the ABD1 gene [7]. ABD1 encodes a 436-amino acid polypeptide. A catalytic domain of Adb1p from residues 110 to 426 suffices for yeast cell growth (8, 9): this segment of Abd1p is homologous to the methyltransferase catalytic domain of the vaccinia virus capping enzyme [7]. A key distinction between the yeast and vaccinia virus cap methyltransferases is their physical linkage, or lack thereof, to the other cap-forming enzymes. The vaccinia virus methyltransferase active site is encoded within the same polypeptide as the triphosphatase and guanylyltransferase, whereas the yeast methyltransferase is a monomeric protein that is not associated with the other capping activities during fractionation of yeast extracts [7]. Mutational analyses of the yeast and vaccinia cap methyltransferases have identified conserved residues that are critical for cap methylation [8, 9, 46]. In the case of Abd1p, mutations that abolished methyltransferase activity in vitro were lethal in vivo.
A putative cap methyltransferase from C. elegans was identified on phylogenetic grounds [9]. An alignment of the sequence of the predicted 402-amino acid C. elegans proteins (Genbank accession Z81038) with the yeast cap methyltransferase Abd1p is shown in FIG. 5. Although it remains to be demonstrated that the nematode protein has cap methyltransferase activity, the extensive sequence conservation suggested that other metazoans might also encode homologues of Abd1p. A human cDNA that encodes a bona fide cap methyltransferase has been identified and a physical and biochemical characterization of the recombinant human cap methyltransferase (Hcm1p) produced in bacteria was conducted. A functional C-terminal catalytic domain of Hem1p was defined by deletion analysis [22].
Interaction of the Cellular Capping Apparatus with the Phosphorylated CTD of RNA Polymerase II. Cap formation in eukaryotic cells in vivo is targeted to the nascent chains synthesized by RNA polymerase II (pol II). A solution to the problem of how pol II transcripts are specifically singled out for capping has been described whereby the cellular capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of pol II [16-18, 47, 48]. The CTD, which is unique to pol II, consists of a tandem array of a heptapeptide repeat with the consenus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The mammalian pol II large subunit has 52 tandem repeats whereas the S. cerevisiae subunit has 27 copies. The pol II largest subunit exists in two forms, a nonphosphorylated IIA form and a phosphorylated IIO form, which are interconvertible and functionally distinct. In vivo, the pol IIO enzyme contains as many as 50 phosphorylated amino acids (primarily phosphoserine) within the CTD. During transcription initiation, pol IIA is recruited to the DNA template by the general transcription factors. The pol IIA CTD undergoes extensive phosphorylation and conversion to IIO during the transition from preinitiation complex to stable elongation complex. Several CTD kinase activities have been implicated in CTD hyperphosphorylation, each of which contains a cyclin and cyclin-dependent kinase subunit pair. The cdk7 and cyclin H subunits of the general transcription factor TFIIH catalyze phosphorylation of Ser-5 of the CTD heptapeptide. Other CTD kinases include the cdk8/cyclin C pair found in the pol II holoenzyme, CTDK-I, a heterotrimeric kinase with cdk-like and cyclin-like subunits, and P-TEFb, a regulator of polymerase elongation with a cdc2-like subunit.
The recombinant S. cerevisiae and Sc. pombe guanlyltransferases Ceg1p and Pce1p bind specifically to the phosphorylated form of the CTD [16]. Moreover, recombinant yeast cap methyltransferase Abd1p also binds specifically to CTD-PO4 [16]. Phosphorylation at Ser-5 of the heptad repeat was sufficient to confer guanylyltransferase and methyltransferase binding capacity to the CTD [16]. This analysis has been extended to mammalian capping enzyme where the key finding is that the guanylyltransferase domain Mce1(211-597)p by itself binds to CTD-PO4, whereas the triphosphatase domain Mce1(1-210)p does not [18]. These findings suggest that the mammalian RNA triphosphatase is targeted to the nascent pre-mRNA by virtue of its connection in cis to the guanylyltransferase. The phosphorylation-dependent interaction between guanylyltransferase and the CTD is conserved from yeast to mammals. It is not clear if the structural elements on the yeast and mammalian enzymes that interact with CTD-PO4 are conserved or divergent. Nonetheless, pharmacological interference with the binding of guanylyltransferase or cap methyltransferase to the CTD is a potential mechanism for blocking gene expression in fungi or mammalian cells. Such interference can occur either by direct blocking of capping enzyme/CTD-POP4 binding or indirectly by affecting the phosphorylation state of the CTD.
The prior art is deficient in the lack of methods of screening for a compound that inhibits formation of an organism""s 5xe2x80x2 mRNA cap structure. The present invention fulfills this longstanding need in the art.
The present invention facilitates the discovery of drugs that target an essential aspect of eukaryotic gene expressionxe2x80x94the formation of the mRNA 5xe2x80x2 cap m7GpppN. The underlying principle of the invention is the use of a different strains of a test organism that differ only in the composition or source of the essential cap-forming enzymes. For example, the construction of isogenic yeast strains that derive all their capping activities from fungal sources versus mammalian sources provides the basis to identify molecules that specifically target the fungal capping apparatus.
The methods disclosed herein test a battery of candidate molecules for their ability to selectively impair the growth of a strain containing capping enzymes differing in composition or source. In a simple embodiment of the invention, this would entail the local application of an array of candidate molecules to agar culture plates that had been inoculated with an engineered yeast strain containing capping enzymes differing in composition or source. The plates are incubated to permit growth of the yeast cells to form a confluent lawn. Growth inhibition by the applied compound is detected as a xe2x80x9chaloxe2x80x9d of no-growth or slow-growth around the site of application. The methods will necessarily detect many compounds that inhibit the growth of all test strains; these are regarded as non-specific to capping and would likely not be pursued. The positive candidates are those that inhibit growth of one strain, but not of an otherwise identical strain containing a capping apparatus that differs in composition or source. For example, molecules that inhibit the growth of yeast cells containing an all-fungal capping apparatus, but have little or no effect on the growth of yeast cells containing the mammalian capping apparatus, would be regarded as promising leads for antifungal drugs. Conversely, molecules that inhibit the growth of cells containing an all-mammalian capping apparatus, but have little or not effect on the growth of cells containing the fungal capping apparatus merit further consideration as specific inhibitors of capping in mammalian cells, with the potential for development as an antineoplastic agent.
The methods of the present invention are also applicable to the identification of potential antiviral agents and antiparasitic agents that specifically target virus-encoded or parasite-encoded cappiny enzymes. In this embodiment of the invention, the growth inhibition screen would include testing the array of candidate molecules against a strain that contains one or more virus-encoded or parasite-encoded capping activities in lieu of the endogenous enzyme(s). Molecules that selectively inhibit growth of the strain bearing the viral or parasite capping enzyme component, but not both of the strain bearing the mammalian capping apparatus, would be regarded as promising leads for further evaluation of antiviral or antiparasitic activity.
The invention is not restricted to the use of fungi as the test organisms. Advances in gene targeting in mammalian cells make it feasible to construct mammalian cell lines in which one or more of the genes encoding cellular capping activities is deleted and replaced by a gene encoding the analogous enzyme from another source. Thus, candidate cap-targeting compounds could be identified by screening in parallel for selective growth inhibition of one of several cell lines differing only in the composition or source of the capping apparatus. Eukaryotic viruses that depend on virus-encoded capping activities can also be developed as the targets for testing of growth inhibition. In this embodiment, a viral gene encoding an essential capping activity would be deleted and replaced by a gene encoding the analogous enzyme from another source. Virus plaque formation on permissive host cells provides an easy visual screen (by plaque number and plaque size) for inhibition of virus replication by candidate agents added to the medium. Agents that selectively inhibit the replication of virus containing capping enzymes from one source, but not from another source, are presumed to do so by selective targeting of cap formation.
The invention is also not restricted to identifying exogenous molecules that target cap formation. Another embodiment facilitates the DNA-based identification of natural or synthetic gene products that inhibit cell growth via intracellular effects on the capping enzymes. In this application of the invention, candidate genes or gene libraries (either natural or synthetic) would be transformed into a test yeast strain, e.g., a strain bearing an all-fungal capping apparatus. The genes in the library are under the control of a regulated promoter (e.g. a GAL promoter) so that their expression can be repressed (in glucose medium) or induced (in galactose medium) by the experimenter. The initial screen selects for library-transformed cells that grow on glucose, but are inhibited on galactose. Plasmids recovered from such cells would be clonally amplified in bacteria and then re-transformed in parallel into yeast strains containing the fungal capping apparatus and strains containing one or both mammalian capping enzymes. Plasmids that elicit galactose-dependent growth inhibition of the strain with the fungal capping system, but do not inhibit the strains with mammalian capping components, are good candidates to encode specific antagonists of fungal cap formation. Sequencing the plasmid encoded gene product will reveal the identity of the presumptive inhibitor. Structure-activity relationships for the gene product can then be delineated by DNA-based mutagenesis.
The growth-inhibiting molecules or genes identified using the methods described in this invention could conceivably target cap formation via a number of distinct mechanisms, including: (i) direct inhibition of the catalytic activity of one of the cap-forming enzymes by the identified molecule or a metabolite thereof; (ii) interference with protein-protein interactions required for in vivo function of one of the cap-forming enzymes; (iii) alterations in the level of available substrates for cap formation (e.g., GTP and AdoMet) or the level of endogenous inhibitors of cap formation (e.g., AdoHcy); and (iv) the synthesis of abnormal cap structures in the presence of the growth-inhibiting molecule (or metabolites thereof) that effectively xe2x80x9cpoisonxe2x80x9d cellular transactions dependent on the RNA cap.
An advantage of the present invention is that it is geared to detect specific targeting of capping enzymes in vivo based on differences in their composition or origin, without bias with respect to the mechanism of inhibition. Once candidate molecules are identified, further testing of growth inhibition of isogenic strains differing in one component of the capping system can reveal which enzyme is targeted. Further mechanistic studies can ensue using purified capping enzymes from the relevant sources.
The invention also encompasses an in vitro screen to identify candidate inhibitors of the catalytic activity of the fungal RNA 5xe2x80x2 triphosphatase. The method exploits the fact that the yeast RNA triphosphatase Cet1p has a vigorous ATPase activity that depends on either manganese or cobalt as the divalent cation cofactor [12]. The method is simple quantitative, and adaptable to a colorimetric detection assay that is suited to high-throughput screening for inhibitors.